This invention relates to the detection and quantification of serum enzymes in living organisms. The term, serum enzyme, as used herein is meant to include those enzymes occurring normally in the serum, but whose concentration will depend upon or be affected by some pathological condition in the organism, as well as those enzymes not detected in the serum of a normal organism but whose concentration increases under pathological conditions. More particularly, the invention relates to the detection and quantification of the serum enzyme Creatine Kinase (EC2.7.32 International Union of Biochemistry), commonly known as Creatine Phosphokinase or CPK (referred to as CPK hereinafter). The invention teaches the detection of this enzyme in dried samples contained in a porous carrier such as filter paper.
The sample is conveniently obtained from any fluid derived from an organic source such as blood, plasma, serum, spinal fluid, organ extracts, tissue fluid, eye tears, body secretions, tissue cultures etc. Studies in the inventor's laboratory and by others have shown the substrate-carried dried sample to be stable for at least one month under normal ambient conditions.
CPK determination is extremely valuable in the detection of neuromuscular disorders (Munsat et al., Journal of the American Medical Association, 226-13, Page 1536, Dec. 24, 1973). The measurement of CPK is also of significant value in the diagnosis of myocardial infarction. There is also growing evidence of abnormal levels of CPK present in many cases of mental disorders. Munsat has further shown that abnormally high CPK levels are present in many carriers of Duchenne Muscular Dystrophy (DMD) and tend to be higher in infancy and adolescence.
Detection of preclinical cases of neuromuscular disorders will facilitate a program of early treatment which can result in prolonged mobility. (Demos, Early Diagnosis and Treatment of Rapidly Developing Duchenne DeBologne-type Myopathy (Type DDB 1); Am. Jour.Phys.Med. 50-6, page 271, 1971). Demos has demonstrated that CPK is already present in very high amounts in newborn victims of Duchenne Muscular Dystrophy. Duchenne Muscular Dystrophy occurs in a frequency of about 1 in 3500 male births with a similar ratio for carriers (females). (Morton and Chung, Formal Genetics of Muscular Dystrophy, Am. Jour.Hum. Gen. 11-4, page 360, Dec., 1959). It also appears that the disease has a mutation rate of about 35%. The method and reagents described herein have been shown to be capable of detecting Duchenne muscular dystrophy preclinically and have also been shown to be capable of detecting the CPK elevations associated with carriers of this disease.
The methods and reagents of the invention are also useful in detecting abnormal levels of CPK in hamsters and pigs. In the 14:6 strain of the Golden Syrian Hamster, this method is extremely valuable as these are cardio-myopathic animals. As such, a non-destructive CPK test (and this is the first such test developed) is invaluable for monitoring the effectiveness of treatment of these animals for cardio and muscular disease using various chemical compounds. In pigs, CPK has been shown to be elevated to significant levels in animals affected by Porcine Stress Syndrom, a disease which causes poor quality pork and substantial losses of pigs due to death in shipment to market.